Perturbation of intracellular calcium homeostasis, increased levels of oxidative stress, and inflammatory mechanisms resulting in excitatory toxicity and neuronal death have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD) (Ito et al. 1994, Putney, 2000; Yoo et al., 2000; Sheehan et al., 1997; De Luigi et al., 2001; Anderson et al., 2001; Remarque et al., 2001). A number of AD-associated abnormalities in intracellular Ca2+ levels and other cellular processes have derived from studies using bradykinin as a stimulus. As a potent inflammation mediator, bradykinin (BK) is produced by brain and peripheral cells under patho-physiological conditions such as trauma, stroke, pain ischemia, and asthma (Regoli et al., 1993; Bockmann & Paegelow, 2000; Ellis et al., 1989; Kamiya et al., 1993). By acting on the B2 bradykinin receptor (BK2bR), a G-protein-coupled receptor, BK triggers phosphatidylinositol (PI) turnover through activity of phospholipase C (PLC), which in turn produces inositol 1, 4, 5-trisphospate (IP3) that increases intracellular Ca2+ release from the IP3-sensitive stores (Noda et al., 1996; Venema et al., 1998; Wassdal et al., 1999; Cruzblanca et al., 1998; Ricupero et al., 1997; Pascale et al., 1999). Through the same pathway, BK also triggers production of other proinflammatory cytokines through activation of mitogen-activated protein (MAP) kinases (Hayashi et al., 2000; Schwaninger et al., 1999; Phagoo et al., 2001). Enhanced elevation of intracellular Ca2+ levels has been found in AD brains as well as in AD peripheral cells in response to stimulation of bradykinin and inactivation of K+ channels (Etcheberrigaray et al., 1993, 1998; Hirashima et al., 1996; Gibson et al., 1996(a)).
Stimulation of PLC subsequent to BK2bR activation also leads to production of diacylglycerol which, along with increased intracellular Ca2+, activates protein kinase C (PKC) isoforms. The BK-activated PLC/phospholipids-Ca2+/PKC cascade interacts with the Ras/Raf signaling pathway, which activates extracellular signal-regulated kinases 1/2 (Erk 1 and Erk2, which are referred to together as “Erk1/2”), a subtype of the MAP kinase family (Berridge, 1984; BAssa et al., 1999; Hayashi et al., 2000; Blaukat et al., 2000, Zhao et al. Neurobiology of Disease 11, 166-183, 2002). Erk1/2 receives signals from multiple signal transduction pathways and leads to cellular proliferation and differentiation by regulation of gene expression through a number of transcriptional factors, including AP-1, NF-κB, and cyclic AMP-responsive element binding protein (CREB). By acting as one of the major kinases, Erk2 phosphorylates tau at multiple serine/threonine sites including Ser-262 and Ser-356 (Reynolds et al., 1993; Lu et al., 1993). In addition, PKC-activated MAP kinase and BK receptor-associated pathways have been shown to regulate formation and secretion of the soluble form of amyloid precursor protein (sAPP) by different laboratories (Desdouits-Magnen et al., 1998; Gasparini et al., 2001; Nitsch et al., 1994, 1995, 1996, 1998). These findings have suggested the possibility that BK-associated sAPP processing may be linked to the PKC-MAP kinase pathway. On the other hand, pathological conditions such as viral infections, increased oxidative stress, aberrant expression of APP, and exposure to APβ cause activation of MAP kinase (Rodems & Spector, 1998; McDonald et al., 1998; Ekinci et al., 1999; Grant et al., 1999) and enhanced tau phosphorylation (Greenberg et al., 1994; Ekinci & Shea, 1999; Knowles et al., 1999). These effects implicate derangement of a MAP kinase signaling pathway(s) in the pathogenesis of AD.
Mitogen-activated protein kinases (such as Erk1 and Erk2) regulate phosphorylation of the microtubule associated protein tau and processing of the amyloid protein β, both events critical to the pathophysiology of Azheimer's disease. Enhanced and prolonged Erk1/2 phosphorylation in response to bradykinin has been detected in fibroblasts of both familial and sporadic Alzheimer's disease, but not age-matched controls (Zhao et al. Neurobiology of Disease 11, 166-183, 2002). Sustained Erk1/2 phosphorylation induced by bradykinin has previously been found in Alzheimer's disease fibroblasts and is the subject of WO 02/067764, which is herein incorporated by reference in its entirety.
There exists a need for highly sensitive and highly specific tests to diagnose Alzheimer's disease and to screen for compounds useful in the treatment and prevention of Alzheimer's disease. The present inventors have identified, for the first time, unique Alzheimer's disease-specific molecular biomarkers useful for the diagnosis of Alzheimer's disease in a highly sensitive and highly specific manner compared to previously known diagnostic tests. Thus, the unique Alzheimer's disease-specific molecular biomarkers disclosed herein serve as the basis for diagnostic methods having a high degree of sensitivity and specificity for the detection and diagnosis of Alzheimer's disease. The unique Alzheimer's disease-specific molecular biomarkers of the present invention are also useful in screening methods to identify compounds which may be used as therapeutic agents in the treatment and prevention of Alzheimer's disease.